Growth prior to thermogenesis for a quick fledging of Adélie penguin chicks (Pygoscelis adeliae).

The evolutionary trade-off between tissue growth and mature function restricts the post natal development of polar birds. The present study uses an original integrative approach as it includes gene expression, plus biochemical and physiological analysis to investigate how Adélie penguin chicks achieve a rapid growth despite the energetic constraints linked to the cold and the very short breeding season in Antarctica. In pectoralis muscle, the main thermogenic tissue in birds, our data show that the transition from ectothermy to endothermy on Day 15 post- hatching is associated with substantial and coordinated changes in the transcription of key genes. While the early activation of genes controlling cell growth and differentiation (avGHR, avIGF-1R, T3Rß) is rapidly down-regulated after hatching, the global increase in the relative expression of genes involved in thermoregulation (avUCP, avANT, avLPL) and transcriptional regulation (avPGC1α, avT3Rß) underlie the muscular acquisition of oxidative metabolism. Adélie chicks only become real endotherms at 15 days of age with the development of an oxidative muscle phenotype and the ability to shiver efficiently. The persistent muscular expression of IGF-1 throughout growth probably acts as a local mediator to adjust muscle size and its oxidative capacity to anticipate the new physiological demands of future Dives in cold water. The up-regulation of T3Rß mRNA levels suggests that circulating T3 may play an important role in the late maturation of skeletal muscle by reinforcing, at least in part, the paracrine action of IGF-1. From day 30, the metabolic shift from mixed substrate to lipid metabolism, with the markedly increased mRNA levels of muscle avLPL, avANT and avUCP, suggests the late development of a fatty acid-enhanced muscle non-shivering thermogenesis mechanism. This molecular control is the key to this finely-tuned strategy by which the Adélie penguin chick successfully heads for the sea on schedule.

Ontogeny of muscle bioenergetics in Adelie penguin chicks (Pygoscelis adeliae).

The ontogeny of pectoralis muscle bioenergetics was studied in growing Adélie penguin chicks during the first month after hatching and compared with adults using permeabilized fibers and isolated mitochondria. With pyruvate-malate-succinate or palmitoyl-carnitine as substrates, permeabilized fiber respiration markedly increased during chick growth (3-fold) and further rose in adults (1.4-fold). Several markers of muscle fiber oxidative activity (cytochrome oxidase, citrate synthase, hydroxyl-acyl-CoA dehydrogenase) increased 6- to 19-fold with age together with large rises in intermyofibrillar (IMF) and subsarcolemmal (SS) mitochondrial content (3- to 5-fold) and oxidative activities (1.5- to 2.4-fold). The proportion of IMF relative to SS mitochondria increased with chick age but markedly dropped in adults. Differences in oxidative activity between mitochondrial fractions were reduced in adults compared with hatched chicks. Extrapolation of mitochondrial to muscle respirations revealed similar figures with isolated mitochondria and permeabilized fibers with carbohydrate-derived but not with lipid-derived substrates, suggesting diffusion limitations of lipid substrates with permeabilized fibers. Two immunoreactive fusion proteins, mitofusin 2 (Mfn2) and optic atrophy 1 (OPA1), were detected by Western blots on mitochondrial extracts and their relative abundance increased with age. Muscle fiber respiration was positively related with Mfn2 and OPA1 relative abundance. Present data showed by two complementary techniques large ontogenic increases in muscle oxidative activity that may enable birds to face thermal emancipation and growth in childhood and marine life in adulthood. The concomitant rise in mitochondrial fusion protein abundance suggests a role of mitochondrial networks in the skeletal muscle processes of bioenergetics that enable penguins to overcome harsh environmental constraints.

Diatom-specific highly branched isoprenoids as biomarkers in Antarctic consumers.

The structure, functioning and dynamics of polar marine ecosystems are strongly influenced by the extent of sea ice. Ice algae and pelagic phytoplankton represent the primary sources of nutrition for higher trophic-level organisms in seasonally ice-covered areas, but their relative contributions to polar marine consumers remain largely unexplored. Here, we investigated the potential of diatom-specific lipid markers and highly branched isoprenoids (HBIs) for estimating the importance of these two carbon pools in an Antarctic pelagic ecosystem. Using GC-MS analysis, we studied HBI biomarkers in key marine species over three years in Adélie Land, Antarctica: euphausiids (ice krill Euphausia crystallorophias and Antarctic krill E. superba), fish (bald notothens Pagothenia borchgrevinki and Antarctic silverfish Pleuragramma antarcticum) and seabirds (Adélie penguins Pygoscelis adeliae, snow petrels Pagodroma nivea and cape petrels Daption capense). This study provides the first evidence of the incorporation of HBI lipids in Antarctic pelagic consumers. Specifically, a di-unsaturated HBI (diene) of sea ice origin was more abundant in ice-associated species than in pelagic species, whereas a tri-unsaturated HBI (triene) of phytoplanktonic origin was more abundant in pelagic species than in ice-associated species. Moreover, the relative abundances of diene and triene in seabird tissues and eggs were higher during a year of good sea ice conditions than in a year of poor ice conditions. In turn, the higher contribution of ice algal derived organic matter to the diet of seabirds was related to earlier breeding and higher breeding success. HBI biomarkers are a promising tool for estimating the contribution of organic matter derived from ice algae in pelagic consumers from Antarctica.

Selective upregulation of lipid metabolism in skeletal muscle of foraging juvenile king penguins: an integrative study.

The passage from shore to marine life of juvenile penguins represents a major energetic challenge to fuel intense and prolonged demands for thermoregulation and locomotion. Some functional changes developed at this crucial step were investigated by comparing pre-fledging king penguins with sea-acclimatized (SA) juveniles (Aptenodytes patagonicus). Transcriptomic analysis of pectoralis muscle biopsies revealed that most genes encoding proteins involved in lipid transport or catabolism were upregulated, while genes involved in carbohydrate metabolism were mostly downregulated in SA birds. Determination of muscle enzymatic activities showed no changes in enzymes involved in the glycolytic pathway, but increased 3-hydroxyacyl-CoA dehydrogenase, an enzyme of the ß-oxidation pathway. The respiratory rates of isolated muscle mitochondria were much higher with a substrate arising from lipid metabolism (palmitoyl-L-carnitine) in SA juveniles than in terrestrial controls, while no difference emerged with a substrate arising from carbohydrate metabolism (pyruvate). In vivo, perfusion of a lipid emulsion induced a fourfold larger thermogenic effect in SA than in control juveniles. The present integrative study shows that fuel selection towards lipid oxidation characterizes penguin acclimatization to marine life. Such acclimatization may involve thyroid hormones through their nuclear beta receptor and nuclear coactivators.

Growing in Antarctica, a challenge for white adipose tissue development in Adelie penguin chicks (Pygoscelis adeliae).

Rapid growth is of crucial importance for Adélie penguin chicks reared during the short Antarctic summer. It partly depends on the rapid ontogenesis of fat stores that are virtually null at hatching but then develop considerably (x40) within a month to constitute both an isolative layer against cold and an energy store to fuel thermogenic and growth processes. The present study was aimed at identifying by RT-PCR the major transcriptional events that chronologically underlie the morphological transformation of adipocyte precursors into mature adipocytes from hatching to 30 days of age. The peak expression of GATA binding protein 3, a marker of preadipocytes, at day 7 posthatch indicates a key proliferation step, possibly in relation to the expression of C/EBPalpha (C/EBPalpha). High plasma total 3,5,3'-triiodo-l-thyronine (T(3)) levels and high levels of growth hormone receptor transcripts at hatching suggested that growth hormone and T(3) play early activating roles to favor proliferation of preadipocyte precursors. Differentiation and growth of preadipocytes may occur around day 15 in connection with increased abundance of transcripts encoding IGF-1, proliferator-activated receptor-gamma, and C/EBPbeta, gradually leading to functional maturation of metabolic features of adipocytes including lipid uptake and storage (lipoprotein lipase, fatty-acid synthase) and late endocrine functions (adiponectin) by day 30. Present results show a close correlation between adipose tissue development and chick biology and a difference in the scheduled expression of regulatory factors controlling adipogenesis compared with in vitro studies using cell lines emphasizing the importance of in vivo approaches.

Involvement of a JAK/STAT pathway inhibitor: cytokine inducible SH2 containing protein in breast cancer.

Cytokines and growth factors are responsible for inducing the expression of suppressor of cytokine signaling (SOCS) and cytokine-inducible SH2 containing (CIS) proteins. SOCS and CIS proteins are negative regulators of the JAK/STAT pathway, and exert their physiological effects by suppressing the tyrosine kinase activity of cytokine receptors and inhibiting STAT activation. Growth hormone (GH) is considered as a true cytokine and its local production directly contributes to tumor progression. In an initial study, we have found that CIS expression is increased in human breast cancer in proliferative areas corresponding to high level of GH synthesis. The results of the study presented here confirm the presence of a negative feed back loop in MCF7 cells stably transfected with the hGH gene (MCF-hGH). Real-time PCR analysis showed that gene expression levels of CIS were increased by 80% in MCF-hGH cells as compared to control cell line. Similarly, we have found that the level of CIS gene expression is increased by 50% in primary cultures of human breast cancer, reinforcing the pathophysiological impact of CIS. We previously demonstrated that increasing levels of transfected CIS resulted in strong activation of the mitogen-activated protein (MAP) kinase pathway. Thus, CIS protein has been hypothesized as acting like an activator of the MAPK pathway and an inhibitor of the differentiated cells functions mediated through the JAK/STAT pathway. In the present study, we demonstrate the role of CIS protein in tumor progression in particular its positive effects on cell proliferation and colony formation.

Proteomic analysis of autocrine/paracrine effects of human growth hormone in human mammary carcinoma cells.

Human growth hormone (hGH) is expressed by mammary epithelial cells and associated with proliferative disorders of the human breast. Our goal is to characterize the paracrine effects of hGH on morphological and functional changes of mammary carcinoma cells using MCF7 cells stably transfected with the hGH gene (MCFhGH). To identify the molecular actors involved in autocrine hGH-induced cell proliferation, we have used a protein chip technology using a commercial antibody microarray. The results enabled us to qualitatively characterize MCF-hGH cell's proteome from a panel of 500 proteins. Statistical analysis of variations in protein levels between the two cell lines did not highlight any significant differences. Thus, we concluded that variations in MCF-hGH proteome are more likely to reside in the activation status rather than drastic variations in the expression level of the 500 spotted proteins. To test this hypothesis, we confronted the protein chip result to the study of the regulation of the transcriptional factor Pax (Paired-box)-5 whose expression was not found to be altered on the protein chip. Surprisingly, we found that autocrine production of hGH in MCF7 cells was associated with a strong nuclear accumulation of Pax5 in a JAK2-dependent manner associated with an increase in Pax5-DNA binding activity. Our work indicates that subtle changes mediated by Pax5 are responsible for autocrine hGH-induced cell proliferation.

C620R mutation of the murine ret proto-oncogene: loss of function effect in homozygotes and possible gain of function effect in heterozygotes.

Germline RET mutations are responsible for different inherited disorders: Hirschsprung disease (congenital aganglionic megacolon), caused by loss of function mutations, familial medullary thyroid carcinoma and multiple endocrine neoplasia type 2, caused by gain of function mutations. Intriguingly, some RET mutations, including C620R, are associated with both types of diseases. To investigate the dual role of such RET mutations, a mouse model with a targeted mutation ret(C620R) was generated. ret(C620R/C620R) offspring die during the first postnatal day, and show kidney agenesis and intestinal aganglionosis. Decreased outgrowth of the Ret-positive cells was observed in ret(C620R/C620R) neuronal cell cultures, which is suggestive of an impaired migration, proliferation or survival of the Ret-expressing cells. Electronmicroscopy revealed the absence of membrane-bound Ret in ret(C620R/C620R) cells as compared to ret(+/+) and ret(+/C620R) cells. On the other hand, aged ret(+/C620R) mice develop precancerous lesions in the adrenal gland or in the thyroid. Our results suggest that the ret(C620R) mutation has a loss of function effect in homozygotes and exhibits a dominant gain of function effect with low penetrance causing hyperplasia in heterozygotes.

Electrophysiological characterization of left ventricular myocytes from obese Sprague-Dawley rat.

OBJECTIVE: Obesity is a complex multifactorial disease that is often associated with cardiac arrhythmias. Various animal models have been used extensively to study the effects of obesity on physiological functions, but, to our knowledge, no study related to ionic membrane currents has been performed on isolated cardiac myocytes. Therefore, we examined the electrophysiological characteristics of four ionic currents from isolated left ventricular myocytes of a high-energy (HE)-induced obesity rat model. RESEARCH METHODS AND PROCEDURES: Male Sprague-Dawley rats were fed with either a control diet or a diet containing 33% kcal as fat (HE) for 14 weeks starting at 6 weeks of age. Voltage-clamp experiments were performed on ventricular myocytes. Leptin receptor (ObR) expression was measured using ObR enzyme-linked immunosorbent assay. RESULTS: In the HE group, rats designated as obese did not develop a cardiac hypertrophy, either at the organ level or at the cellular level. Densities and kinetics of the L-type calcium current, the transient outward potassium current, the delayed rectifier potassium current, and the sodium-calcium exchange current (I(NCX)) were not significantly different between control and obese rats. A down-regulation of ObR expression was evidenced in the heart of obese rats compared with controls. Acute exposure (5 minutes) of leptin (100 nM) did not induce a significant modification in the current densities either in control or in obese rats, except for I(NCX) density measured in control rats. DISCUSSION: The absence of effect of leptin on I(NCX) in obese rats could be a potential arrhythmogenic substrate in obesity.

Autocrine growth hormone prevents lactogenic differentiation of mouse mammary epithelial cells.

We have examined the expression, postnatal ontogeny, and localization of mouse GH (mGH) and its relative expression during pregnancy, lactation, and weaning in the mouse. mGH mRNA and protein was expressed predominantly in the epithelial component of the mammary gland, and maximal expression was observed during the pubertal period. Autocrine mGH expression dramatically decreased during late pregnancy and lactation. Concordantly, autocrine mGH expression is repressed during forced differentiation of mouse HC11 mammary epithelial cells in culture. Forced expression of mGH in HC11 cells abrogated lactogenic differentiation as indicated by reduced expression of beta-casein and reduced expression and loss of lateral epithelial localization of E-cadherin. Forced expression of mGH in mouse mammary epithelial cells increased cell survival and proliferation and consequently increased the size of mammary acinar-like structures formed in three-dimensional Matrigel. Thus, autocrine mGH expression in the mouse mammary epithelial cell is maximal at puberty and prevents mammary epithelial cell differentiation. Autocrine GH will therefore participate in mammary morphogenic processes at puberty.

Growth hormone activity in mitochondria depends on GH receptor Box 1 and involves caveolar pathway targeting.

Growth hormone (GH) binding to its receptor (GHR) initiates GH-dependent signal transduction and internalization pathways to generate the biological effects. The precise role and way of action of GH on mitochondrial function are not yet fully understood. We show here that GH can stimulate cellular oxygen consumption in CHO cells transfected with cDNA coding for the full-length GHR. By using different GHR cDNA constructs, we succeeded in determining the different parts of the GHR implicated in the mitochondrial response to GH. Polarography and two-photon excitation fluorescence microscopy analysis showed that the Box 1 of the GHR intracellular domain was required for an activation of the mitochondrial respiration in response to a GH exposure. However, confocal laser scanning microscopy demonstrated that cells lacking the GHR Box 1 could efficiently internalize the hormone. We demonstrated that internalization mediated either by clathrin-coated pits or by caveolae was able to regulate GH mitochondrial effect: these two pathways are both essential to obtain the GH stimulatory action on mitochondrial function. Moreover, electron microscopic and biochemical approaches allowed us to identify the caveolar pathway as essential for targeting GH and GHR to mitochondria.

Cloning, expression and regulation of chicken ovalbumin upstream promoter transcription factors (COUP-TFII and EAR-2) in the rat anterior pituitary gland.

Chicken ovalbumin upstream promoter transcription factors (COUP-TF)-II (NR2F2) and EAR-2 (NR2F6) are structurally related orphan members of the nuclear receptors superfamily. There are growing evidences that these factors play important roles during processes of differentiation and proliferation of several tissues. To better understand their role in the differentiated adult rat pituitary gland, we cloned COUP-TFII and EAR-2 cDNAs from an anterior pituitary cDNA library. Subsequently, we raised and characterized specific antibodies to the N-terminal domain of both nuclear receptors. We next examined their cellular and subcellular distribution in the pituitary gland and determined their regulation during pregnancy. COUP-TFII and EAR-2 pituitary genes display, respectively, 90 and 100% homologies with their human and mouse homologues. Cellular expression of both nuclear receptors was mainly detected in the lactotropes of male and female rats, with a prominent distribution in the nuclear compartment for EAR-2, and interestingly both proteins were significantly upregulated in pituitaries of pregnant vs. cycling female rats. Thus, our results have characterized cloning of rat pituitary COUP-TFII and EAR-2 genes, demonstrated that they are both specifically expressed in lactotropes, and strongly suggested that they may play an important role in modulating prolactin (PRL) gene expression during pregnancy.

Growth hormone receptor expression in atrophying muscle fibers of rats.

Biological actions of GH on muscle growth and metabolism are mediated through specific trans-membrane receptors. The aim of this study was to determine GH receptor (GHR) mRNA expression in muscle atrophy. GHR gene expression in the rat was investigated by in situ hybridization and RT-PCR in slow-twitch oxidative muscle [soleus (SOL)] and fast-twitch glycolytic muscle [extensor digitorum longus (EDL)] after 7 and 35 d of hindlimb unloading. In control rats, the RT-PCR mRNAs levels of GHR were greater (+34%) in EDL compared with SOL. At single fiber level, relative expression of GHR mRNA increases in the following order: IIb>IIa>I. After hindlimb unloading, GHR expression significantly increased in atrophied SOL muscle after 7 (+170%) and 35 (+220%) d, whereas no significant alterations appeared in the EDL muscle. At the individual fiber level, in situ hybridization demonstrated this increase was accounted for by an increase in type I fiber expression of GHR transcripts. This increase was also seen in the EDL, but the low content of type I fibers in EDL resulted in a nonsignificant increase in GHR transcript content. The present data suggest that muscle atrophy is associated with a muscle fiber type-specific GHR mRNA up-regulation mechanism that helps protect atrophying fibers in EDL but might be part of an attempt to repair in SOL.

Nuclear translocation and retention of growth hormone.

We have previously demonstrated that GH is subject to rapid receptor-dependent nuclear translocation. Here, we examine the importance of ligand activation of the GH-receptor (GHR)-associated Janus kinase (JAK) 2 and receptor dimerization for hormone internalization and nuclear translocation by use of cells stably transfected with cDNA for the GHR. Staurosporine and herbimycin A treatment of cells did not affect the ability of GH to internalize but resulted in increased nuclear accumulation of hormone. Similarly, receptor mutations, which prevent the association and activation of JAK2, did not affect the ability of the hormone to internalize or translocate to the nucleus but resulted in increased nuclear accumulation of GH. These results were observed both by nuclear isolation and confocal laser scanning microscopy. Staurosporine treatment of cells in which human GH (hGH) was targeted to the cytoplasm (removal of secretion sequence) or to the nucleus (addition of the nuclear localization sequence of SV40 large T antigen) resulted in preferential accumulation of hGH in the nucleus. We further investigated the requirement of receptor dimerization for GH nuclear translocation using the non-receptor-dimerizing hGH antagonist, hGH-G120R, conjugated to fluorescein isothiocyanate. Confocal laser scanning microscopy demonstrated efficient internalization of both hGH and hGH-G120R but lack of nuclear translocation of hGH-G120R. Thus, we conclude that activation of JAK2 kinase and the subsequent tyrosine phosphorylation is not required for nuclear translocation of GH but is pivotal for the removal of the hormone from the nucleus, and that GH translocates into the nucleus in a GHR dimerized-dependent fashion.